Cold Spring Harbor Protocols

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ISSN / EISSN : 1940-3402 / 1559-6095
Published by: Cold Spring Harbor Laboratory (10.1101)
Total articles ≅ 9,854
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Edward A. Greenfield
Cold Spring Harbor Protocols, Volume 2022; https://doi.org/10.1101/pdb.prot103085

Abstract:
Flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to internal cellular proteins, but the cells must be permeabilized before the hybridoma supernatants are applied. In using this technique, useful controls are positive and negative cell lines with primary and secondary antibodies as well as positive and negative cell lines with secondary antibody alone.
Edward A. Greenfield
Cold Spring Harbor Protocols, Volume 2022; https://doi.org/10.1101/pdb.prot103077

Abstract:
If the antigen of interest is a cell-surface protein, flow cytometry or fluorescence-activated cell sorting (FACS) can be used to identify hybridomas secreting monoclonal antibodies to these proteins. Two alternative protocols are presented here—staining in individual tubes and staining in 96-well plates.
Scott J. Rodig
Cold Spring Harbor Protocols, Volume 2022; https://doi.org/10.1101/pdb.prot099648

Abstract:
Staining yeast cells for the presence and location of antigens is particularly challenging. They are small, making the resolution of any antigen difficult; they have a thick cell wall that antibodies cannot penetrate and that is difficult to remove; and they grow in suspension, making handling difficult. In addition, background problems can be especially severe, particularly with polyclonal antibodies, because many antisera contain antibodies to yeast cell wall components. In this protocol, yeast cells are treated with paraformaldehyde, the cell wall is removed by enzymic digestion, and the spheroplasts are attached to poly-l-lysine-coated slides. After cell lysis, the cells are ready to be stained as per normal. Except in unusual circumstances, the detection reagent should be fluorochrome-labeled.
Scott J. Rodig
Cold Spring Harbor Protocols, Volume 2022; https://doi.org/10.1101/pdb.top099606

Abstract:
When labeled antibodies are used to detect antigens in cells or tissues, several characteristics of an antigen can be readily determined. Most importantly, cell staining will show both the presence and subcellular localization of an antigen. Double-labeling techniques permit the simultaneous detection of two antigens, allowing comparisons of the relative distribution of different antigens. Many cell-staining methods can also be used in conjunction with conventional histological stains and autoradiographic methods to compare the localization of the antigen with other markers. Cell staining can also be used in pathology studies for determining such variables as the type of infectious organism, the progenitor of a neoplastic cell, or the presence of an inflammatory response. With certain modifications, the procedure can be used to purify cells on the basis of the antigenic composition of their cell surface. Cell staining is a versatile technique and, if the antigen is highly localized, can detect as few as a thousand antigen molecules in a cell or tissue. In some circumstances, cell staining may also be used to determine the approximate concentration of an antigen. Improvements in antibody labeling methods, microscopes, cameras, and image analyzers are rapidly extending the sensitivity of cell-staining procedures and are making these techniques more quantitative. Even without these improvements, cell staining can yield important qualitative and semiquantitative data. This introduction describes protocols for cell staining techniques and includes a discussion of major constraints, antibody selection, and troubleshooting.
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